Thorax Online First, published on February 3, 2017 as 10.1136/thoraxjnl-2016-209501 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from ORIGINAL ARTICLE α-Tocopherol transfer protein mediates protective hypercapnia in murine ventilator-induced lung injury Gail Otulakowski,1 Doreen Engelberts,1 Hajime Arima,1,2,3 Hiroyuki Hirate,1,2,3 Hülya Bayir,4,5 Martin Post,1 Brian P Kavanagh1,2,6

▸ Additional material is ABSTRACT published online only. To view Rationale Hypercapnia is common in mechanically Key messages please visit the journal online ‘ (http://dx.doi.org/10.1136/ ventilated patients. Experimentally, therapeutic thoraxjnl-2016-209501). hypercapnia’ can protect, but it can also cause harm, depending on the mechanism of injury. Hypercapnia 1Physiology and Experimental What is the key question? Medicine, Hospital for Sick suppresses multiple signalling pathways. Previous ▸ Can molecular mechanisms induced by Children, Toronto, Canada investigations have examined mechanisms that were hypercapnia during mechanical ventilation 2 Department of Critical Care known a priori, but only a limited number of pathways, reveal potential therapeutic pathways for lung Medicine, Hospital for Sick each suppressed by CO2, have been reported. injury? Children, Toronto, Canada ▸ 3Department of Anesthesiology Objective Because of the complexity and Does hypercapnia protect against lung injury by and Intensive Care Medicine, interdependence of processes in acute lung injury, this activating protective genes? Nagoya City University study sought to fill in knowledge gaps using an Graduate School of Medical unbiased screen, aiming to identify a specifically What is the bottom line? Sciences, Nagoya, Japan ▸ Inspired CO upregulates a transport protein 4 upregulated pathway. 2 Department of Environmental (α-tocopherol transfer protein); this mobilises and Occupational Health, Methods and results Using genome-wide gene Center for Free Radical and expression analysis in a mouse model of ventilator- vitamin E in the lung which inhibits synthesis of Antioxidant Health, University induced lung injury, we discovered a previously a pathogenic chemoattractant, leukotriene B4. of Pittsburgh, Pittsburgh, unsuspected mechanism by which CO can protect Pennsylvania, USA 2 Why read on? 5 against injury: induction of the transporter protein for Department of Critical Care ▸ Experimental elevation of CO2 can protect (or Medicine, Safar Center for α-tocopherol, α-tocopherol transfer protein (αTTP). α cause harm); this new induced mechanism of Resuscitation Research, Pulmonary TTP was induced by inspired CO2 in two in protection from CO provides new potentially University of Pittsburgh, vivo murine models of ventilator-induced lung injury; the 2 Pittsburgh, Pennsylvania, USA testable therapies for lung injury. 6 level of αTTP expression correlated with degree of lung Department of Anesthesia, α fi University of Toronto, Toronto, protection; and, absence of the TTP gene signi cantly http://thorax.bmj.com/ Canada reduced the protective effects of CO2. α-Tocopherol is a potent antioxidant and hypercapnia increased lung Correspondence to α-tocopherol in wild-type mice, but this did not alter fl Dr Brian P Kavanagh, elevated CO2 inhibits expression of in ammatory superoxide generation or expression of NRF2-dependent cytokines,5 activation of nuclear factor κB Department of Critical Care −/− Medicine, Hospital for Sick antioxidant response genes in wild-type or in αTTP (NFκB)8 and sheddase (a disintegrin and metallo- Children, 555 University mice. In concordance with a regulatory role for protease 17; ADAM17)6 signalling, and func- Avenue, Toronto, Ontario, α-tocopherol in lipid mediator synthesis, hypercapnia tional protein expression (eg, endocytosis of ion Canada M5G 1X8; attenuated 5-lipoxygenase activity and this was channels).7 However, these observations are [email protected] on September 30, 2021 by guest. Protected copyright. dependent on the presence of αTTP. limited in scope because all were based on injury α Received 28 September 2016 Conclusions Inspired CO2 upregulates TTP which processes known a priori. Also, many of the Revised 6 January 2017 increases lung α-tocopherol levels and inhibits synthesis mechanisms are pivotal biological processes and Accepted 9 January 2017 of a pathogenic chemoattractant. thus have limited translational potential (eg, inhibition of NFκB). We sought to address this knowledge gap using an unbiased screen. We chose a microarray approach INTRODUCTION because the technology is mature, and because regu- Hypercapnia is common in mechanically ventilated lation at the level of gene expression would be a patients with injured lungs, where tidal volume is proximal and fundamental step in most identified deliberately lowered to lessen the risk of ventilator- signalling or synthetic processes. Microarray ana- induced lung injury (VILI).1 However, accumulat- lyses have led to identification of key molecular 9 ing evidence indicates that CO2 can have potent pathways in VILI and of genes regulated by hyper- effects in injured tissue that are independent of capnia in neonatal lung development,10 and provide changes in mechanical ventilation.2 Depending on the possibility of identifying a specifically upregu- the context, these effects may be protective, for lated mechanistic pathway. Hypercapnia-dependent To cite: Otulakowski G, example, attenuating reperfusion injury,3 acute transcriptional responses have been recently Engelberts D, Arima H, et al. 4 56 11 Thorax Published Online sepsis or ventilator-induced injury; or, injurious, reviewed. While not fully characterised, they 12 First: [please include Day for example, impairing alveolar fluid clearance.7 include inhibition of NFκB and hypoxia-inducible Month Year] doi:10.1136/ Several molecular mechanisms of hypercapnic factor 1α (HIF-1α)13 responses, and activation of thoraxjnl-2016-209501 action have been described. For example, CREB14 and FoxO3a.15

Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 1 Copyright Article author (or their employer) 2017. Produced by BMJ Publishing Group Ltd (& BTS) under licence. Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from

We utilised a mouse model of VILI in which addition of CO2 Inflammatory mediators to the inspired gas protects against injury.5 We found that hyper- Myeloperoxidase (MPO) was measured in lung homogenates capnia activated few genes; however, hypercapnia was protective using o-dianisidine dihydrochloride and H2O2 as substrates. in VILI, and here it increased the expression of α-tocopherol Cytokines in bronchoalveolar (BAL) fluid were quantitated transfer protein (αTTP). We describe how increased expression using a Milliplex mouse Cytokine Immunoassay Kit (Millipore, of αTTP may contribute to the protection afforded by hyper- Billerica, Massachusetts, USA). Eicosanoids in BAL were quanti- capnia in this multifaceted in vivo model. tated by liquid chromatography (LC)–tandem mass spectrometry (MS), with cysteinyl leukotrienes (CysLTs) measured by enzyme METHODS immunoassay (Enzo Life Sciences, Farmingdale, New York, Additional details can be found in the online supplementary USA). Superoxide radical in hydroethidine-treated mouse lung data. was detected through quantification of 2-hydroxyethidium using LC-MS-MS analysis. Animal model All animal procedures were approved by the animal care com- Lung tissue α-tocopherol mittee of the Hospital for Sick Children (Toronto, Ontario, Lung tissue α-tocopherol was quantitated using HPLC from Canada) in accordance with the Guidelines of the Canadian homogenates spiked with internal standard δ-tocopherol – Council on Animal Care. C57BL/6J male mice (20 25 g, (Sigma-Aldrich Canada Co., Oakville, Ontario, Canada). Charles River, St Constant, Quebec, Canada) were anesthetised and ventilated: tidal volume (VT) 10 mL/kg, positive Statistics end-expiratory pressure (PEEP) 2.0 cm H2O, frequency 135/ Data are presented as dot plots with mean (for normally distrib- min, FiO2 0.21. Two models were employed. Model-1: mice were randomised to Severe Injury as previously described5 uted data) or median (non-normal distributions) indicated by a horizontal bar. Statistical analyses were calculated with (peak inspiratory pressure 27 cm H2O, VT 35–40 mL/kg, PEEP Sigmaplot V.12.3 (Systat software Inc) using ANOVA for multi- 0cmH2O, frequency 30–35/min) versus continuation of base- group comparisons. Two group comparisons used t tests on nor- line (low VT) ventilation with normocapnia (FiO2 0.75, FiCO2 mally distributed data, and Mann–Whitney U for non-normally 0, balance N2) or hypercapnia (FiO2 0.75, FiCO2 0.12, balance distributed data. p values <0.05 were considered significant. N2) for 3 hours. Model-2: mice were subjected to Moderate Injury (VT 20 mL/kg, PEEP 0 cm H2O, frequency 45/min) for 4 hours, randomised to normocapnia (room air) or hypercapnia RESULTS (FiCO2 0.12, balance room air). Gene expression and hypercapnic protection against ventilator-induced lung injury Genetically modified mice High VT ventilation was used to generate VILI in C57BL/6J −/− 1Far 6 Male knockout (αTTP ) (strain B6.129S4-Ttpa /J, Jackson mice, as previously published; CO2 (hypercapnia) was added to Laboratories, Sacramento, California, USA) and wild-type the inspired gas to protect against VILI in two of five experi- +/+ sibling (αTTP ) controls were subjected to severe injury mental groups: (1) non-ventilated, (2) low VT normocapnia, (3) http://thorax.bmj.com/ (Model-1) and randomised to normocapnia or hypercapnia. low VT hypercapnia, (4) high VT normocapnia, and (5) high VT 6 hypercapnia. Lung injury was significantly greater in high VT Microarray analysis normocapnia compared with all other groups, and hypercapnia 6 We performed gene expression analysis using lung samples from protected against VILI. PaO2, PaCO2 and pH values are given mice included in our previous study,6 ventilated with the Severe in online supplementary table S2. Injury protocol (Model-1). RNA from five groups: (1) non- Microarray analysis of lung samples was used to determine ventilated, (2) low VT normocapnia, (3) low VT hypercapnia, gene expression patterns associated with hypercapnic protec- (4) high V normocapnia, and (5) high V hypercapnia (n=5/ tion. PCA confirmed that whole gene expression profiles were T T on September 30, 2021 by guest. Protected copyright. group non-ventilated and low VT; n=10/group high VT)was similar among samples within each group (figure 1A). Two-way hybridised to Affymetrix (Santa Clara, California, USA) mouse ANOVA analysis of overall gene expression with ventilation and gene 1.0 ST arrays. Primary datasets are accessible through CO2 as factors both showed significant sources of variation, but NCBI GEO (series accession number GSE86229). Data were no significant interaction (figure 1B). High VT ventilation (nor- subjected to robust multichip analysis normalisation; prelimin- mocapnia) changed the expression of 1658 gene probe sets ary principal components analysis (PCA) identified two outliers (contrasted with no ventilation or low VT; fold change 1.5, which were subsequently excluded from downstream analysis. p<0.05), including large increases in inflammatory gene expres- Analysis of changes in gene expression was performed using sion (see online supplementary table S3). Partek Genomics Suite (Partek Inc, St Louis, Michigan, USA), by In high VT ventilation, there were modest changes in gene two-way analysis of variance (ANOVA) using ventilation (non- expression when contrasting normocapnia with hypercapnia. ventilated, high VT,lowVT) and CO2 (normocapnia, hypercap- Hierarchical clustering of this gene set (figure 1C) confirmed nia) as factors, Benjamini and Hochberg false discovery rate 5%, distinct expression patterns, with some overlap in low VT (unin- and fold-change cutoff at 1.5. Overrepresented canonical path- jured) ventilation. A small number of genes were overexpressed ways associated with VT and hypercapnia were identified using in high VT hypercapnia versus high VT normocapnia, and most Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood differentially expressed genes displayed downregulation under City, California, USA). hypercapnia. Twenty-nine independent known genes responding to hypercapnia during high VT ventilation were identified RT-PCR (table 1). We selected 12 candidate genes from this list based on Changes in gene expression were measured using relative quan- plausibly protective effect and regulation at the transcriptional titative real-time PCR by the ddCt method. Primers are shown level, and real time RT-PCR confirmed that nine were regulated in online supplementary table S1. by hypercapnia (gene symbols: Ttpa, Pmvk, Pdgfb, Ereg, Egr2,

2 Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from http://thorax.bmj.com/ on September 30, 2021 by guest. Protected copyright.

Figure 1 Microarray data in murine ventilator-induced lung injury and hypercapnia. (A) Principal component analysis plot for microarray data showing effects of ventilation and hypercapnia. Note: samples 72 and 49, from the high stretch normocapnia group, fell closer to the high stretch hypercapnia cluster than the high stretch normocapnia cluster and were excluded from further analysis. (B) Sources of variation in total gene expression estimated by two-way ANOVA. F ratio (y-axis) for each factor (x-axis) represents the F-statistics for that factor relative to F-statistic for error (noise) for all genes. (C) Cluster analysis showing changes in mRNA abundance during mouse lung ventilation as determined by microarray analysis. Each column represents RNA from a single mouse, and the heatmap shows genes detected by Robust Multichip Average analysis with expression increased (red) or decreased (blue) by 1.5 fold. HS, high stretch; LS, low stretch; HC, hypercapnia; NC, normocapnia; NV, non-ventilated.

Tnc, Gsta1, Ncr1 and Hmox1); three trended as in microarrays, normocapnia (confirmed by RT-PCR; table 1). In addition, the but were not significant (P2ry10, Cckar, Ctgf; table 1). degree of protection (eg, decrement in static compliance) from hypercapnia was proportional to the degree of expression of the Identification of candidate gene αTTP in VILI αTTP mRNA (figure 2). We used a second model of more mod- The most highly upregulated gene, encoding αTTP, was signifi- erate ventilation injury (VT=20 mL/kg; FiO2=0.21; see online fi cantly more expressed in high VT hypercapnia versus high VT supplementary gure S1), in which hypercapnia attenuated Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 3 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from Table 1 List of genes differentially expressed by hypercapnia in severe injury VILI Expression by RTPCR (relative to NV; mean Expression change by microarray ±SEM)

Symbol Gene p Value* (hyper vs normo) Fold change (hyper vs normo) Normo Hyper

Ttpa α-tocopherol transfer protein 7.99E-07 1.74 0.94±0.12 2.11±0.42† Tat tyrosine aminotransferase 8.56E-04 1.71 not tested Pmvk phosphomevalonate kinase 3.80E-05 1.66 4.70±0.70 8.50±1.20‡ P2ry10 purinergic receptor P2Y, G-protein coupled 10 2.33E-03 1.55 1.60±0.16 3.04±0.88 Fat3 FAT tumour suppressor homologue 3 (Drosophila) 5.31E-05 1.53 not tested Zc3h6 zinc finger CCCH type containing 6 1.56E-05 1.53 not tested Cckar cholecystokinin A receptor 2.78E-04 1.52 0.32±0.03 0.48±0.06 Plcxd3 phosphatidylinositol-specific phospholipase C, X domain c 3.35E-08 1.50 not tested Gsta2 glutathione S-transferase, α 2 (Yc2) 1.30E-05 −1.50 not tested Ctps cytidine 5’-triphosphate synthase 3.89E-07 −1.52 not tested Pus7l pseudouridylate synthase 7 homologue (S. cerevisiae)-like 3.67E-06 −1.52 not tested Pdgfb platelet-derived growth factor, B polypeptide 8.12E-06 −1.56 1.08±0.10 0.55±0.06† Scel sciellin 1.16E-03 −1.58 not tested Sh2d4a SH2 domain containing 4A 1.80E-09 −1.58 not tested Ereg epiregulin 8.22E-07 −1.59 5.82±0.95 2.27±0.27‡ Egr2 early growth response 2 1.43E-04 −1.60 9.21±1.80 4.23±0.57‡ Tnc tenascin C 2.94E-04 −1.61 4.21±1.01 1.60±0.28‡ Gsta1 glutathione S-transferase, α 1 (Ya) 1.71E-05 −1.62 6.91±1.33 2.58±0.45† Rnd1 Rho family GTPase 1 1.74E-04 −1.63 not tested Mthfd2 methylenetetrahydrofolate dehydrogenase 2.44E-05 −1.64 not tested Ccl7 chemokine (C-C motif) ligand 7 1.26E-06 −1.65 not tested Ctgf connective tissue growth factor 1.39E-04 −1.67 3.95±0.822 1.95±0.42 Slc26a4 solute carrier family 26, member 4 9.52E-06 −1.75 not tested Timp1 tissue inhibitor of metalloproteinase 1 5.00E-05 −1.82 not tested Ncr1 natural cytotoxicity triggering receptor 1 5.82E-07 −1.82 0.65±0.07 0.26±0.03† Cxcl5 chemokine (C-X-C motif) ligand 5 7.43E-05 −1.88 not tested Spic Spi-C transcription factor (Spi-1/PU.1 related) 1.07E-03 −1.96 not tested Hmox1 heme oxygenase (decycling) 1 6.72E-05 −1.98 12.9±1.99 4.51±0.59

Ccl2 chemokine (C-C motif) ligand 2 2.16E-06 −2.05 not tested http://thorax.bmj.com/ *Adjusted p value, FDR <0.05, Benjamini Hochberg. †p<0.01 vs high stretch normocapnia; N=10/group. ‡p<0.05 vs high stretch normocapnia.

injury (ie, BAL protein, lung MPO), and induced αTTP mRNA (figure 3).

The top canonical pathways associated with gene expression on September 30, 2021 by guest. Protected copyright. from high VT (figure 4A) included inflammation-related signal- ling pathways known to be associated with VILI (NFκB, IL-6, p38 MAPK, HMGB1). A single pathway, the NRF2-mediated oxidative stress response, was shared by high VT and by hyper- capnia (figure 4B); this is a protective response against oxidative 16 and xenobiotic toxicity, and potentially against VILI. High VT increased expression of NRF2 target genes, but these were decreased (not further increased) with hypercapnia (figure 5). αTTP, the most upregulated gene, is a cytosolic protein that shuttles α-tocopherol (vitamin E), an important antioxidant, between cellular membranes. We hypothesised that increased αTTP expression in hypercapnia provides antioxidant protection upstream of the antioxidant NRF2 pathway, thereby limiting its induction. Figure 2 Hypercapnic protection and expression of α-tocopherol transferase protein (αTTP). Linear regression analysis indicating αTTP gene deletion: impact on protection from CO2 correlation between αTTP mRNA expression (RT-PCR) and decrease in To examine the role of αTTP in hypercapnia-mediated protec- static compliance in ventilated mice. Dotted lines indicate 95% CIs. − − tion from VILI, we used knockout (αTTP / ) and wild-type R=−0.73, p<0.001.

4 Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from protection (figure 8B) (as with C57BL/6J mice: table 1, figure 2). In addition, lung tissue α-tocopherol levels were increased +/+ by hypercapnia during high VT ventilation in αTTP sibs − − (figure 8C), while in αTTP / sibs, levels were 20–50 fold less and were not altered by ventilation (figure 8D).

α-Tocopherol and oxidative stress in ventilator-induced lung injury The decrease in NRF2 target gene expression due to hypercap- nia in the C57BL/6J mice (figure 4B) suggested that hypercapnic induction of αTTP may exert antioxidant effects by shuttling α-tocopherol; reduced oxidative stress would then explain the lessened induction of NRF2 target gene expression. If this were true, then in the setting of lung injury, hypercapnia should lower the expression of NRF2 target genes in αTTP+/+ sibs, but − − not in αTTP / mice. Constitutive expression of HMOX1 and − − GSTA1 was highest in lungs of αTTP / (vs αTTP+/+) and lowest in C57BL/6J mice (figure 9A, B), perhaps an adaption to − − low α-tocopherol levels. However, αTTP / and αTTP+/+ mice showed similar changes in HMOX1 and GSTA1 in response to ventilation and hypercapnia (figure 9C, D), indicating that these effects are independent of αTTP expression. To determine whether αTTP protected via an antioxidant mechanism, we measured 2-hydroxyethidium as a unique marker of superoxide generation in mice injected with hydro- − − ethidine; hypercapnia had no impact on αTTP / or αTTP+/+ mice (figure 10A). Similar results were found using multiple markers of oxidative stress (eg, 8-isoprostane, protein carbonyls, and antioxidant capacity assay: data not shown).

α-Tocopherol and eicosanoids in lung injury α-Tocopherol inhibits 5-lipoxygenase (5-LOX),17 and its down- stream products, the leukotriene B4 (LTB4) and cysteinyl leuko- trienes (CysLTs: LTC4, LTD4 and LTE4), are pathogenic in lung injury and ARDS.18 Hypercapnia suppressed the BAL level of − − http://thorax.bmj.com/ LTB4 and CysLTs in αTTP+/+, but not in αTTP / , mice (figure 10B, C). A similar trend was observed for the additional 5-LOX product 5-hydroxyeicosatetraenoic acid (5-HETE) (see online supplementary table S4). There were no differences in levels α Figure 3 Hypercapnic protection and expression of -tocopherol of the eicosanoid precursor arachidonic acid (figure 10D), or in α transferase protein ( TTP) in milder injury. Hypercapnia reduced protein other downstream products (eg, HETEs, prostaglandins or epox- concentration in the bronchoalveolar lavage (BAL) fluid (A), lung tissue yeicosatrienoic acids) (see online supplementary table S4). myeloperoxidase (MPO) activity (B), and increased the tissue expression of αTTP mRNA (C), in a model of milder ventilator-induced lung injury on September 30, 2021 by guest. Protected copyright. DISCUSSION (4 hours, VT=20 mL/kg) (*p<0.05 vs normocapnia, t test). Data from small numbers of non-ventilated mice are presented for illustrative The current study provides evidence of a previously unsuspected purposes but excluded from statistical analysis. mechanism by which CO2 can protect against lung injury: induction of a transporter protein for α-tocopherol, αTTP. Addition of CO2 to the inspired gas increased pulmonary +/+ α sibling (αTTP ) mice subjected to high VT ventilation. All expression of TTP mRNA in different in vivo murine models mice had similar lung compliance at baseline, and similar of VILI; the level αTTP expression correlated with degree of α decreases in compliance over 3 hours during high VT ventilation lung protection; and, absence of the TTP gene abolished the with normocapnia (see online supplementary figure S2A). protective effects of CO2. These data provide new insights into +/+ Hypercapnia attenuated several markers of injury in αTTP the mechanisms of action of CO2 on molecular pathways in − − but not αTTP / mice: compliance (figure 6A, B), lung tissue lung injury beyond previously described multifactorial inhibitory MPO and TNFα mRNA (figure 7A, B), and BAL levels of effects such as NFκB,8 ADAM176 and Na,K-ATPase.7 In con- MCP-1 and KC (figure 7C, D). Also, hypercapnia tended to trast to previously described mechanisms, the current data dem- +/+ decrease BAL IL-6 in αTTP mice only (figure 7E). onstrate that CO2 upregulates expression of αTTP, which increases lung α-tocopherol levels; α-tocopherol can inhibit syn- thesis of the inflammatory mediator leukotrienes, products of Role of αTTP and α-tocopherol in protection with 5-LOX which are pathogenic in acute lung injury.18 hypercapnia Although αTTP was required for hypercapnia’s protective Hypercapnia induced lung tissue expression of αTTP mRNA in effects, we did not find that αTTP knockout (KO) mice injured +/+ αTTP sibs (figure 8A) following high VT ventilation, and more readily than wild-type mice in normocapnic conditions; in greater expression of αTTP mRNA was associated with greater fact, the opposite may be true, since the decrease in static

Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 5 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from Figure 4 Top canonical pathways in response to mechanical ventilation. Differences in mRNAs in lungs of non-ventilated and ventilated mice were identified through microarray analysis and submitted to functional enrichment analysis using Ingenuity Pathway Analysis. Highly significant genes were selected by Fisher’s exact test (at p<0.0001), and the statistical significance of each pathway is presented as negative log (p value). Pathways associated with genes affected by stretch (A) or associated with genes affected by hypercapnia (B) are illustrated. http://thorax.bmj.com/ on September 30, 2021 by guest. Protected copyright.

Figure 5 Relative expression of mRNAs of the antioxidant response pathway in lungs of mechanically ventilated mice. NRF2 target genes heme oxygenase (HMOX1; A) and glutathione-S-transferase (GSTA1; B) were more highly expressed in high stretch normocapnia than all other ventilated groups ; that is, addition of hypercapnia reduced the expression of each during high stretch ventilation (*p<0.01 vs all other groups, one-way ANOVA). compliance in normocapnic KO mice appears slightly less than affinity, other forms of vitamin E, from endosomes to the in wild-type mice (figure 6A,B). This may be due to higher con- plasma membrane in exchange for PIP2; this promotes stitutive levels of other protective mechanisms (eg, NRF2 α-tocopherol secretion into the bloodstream, bound to lipopro- responsive antioxidant genes; figure 9) as a compensatory mech- tein particles.19 Expression of αTTP has been described outside anism in KO animals which have very low levels of the liver (eg, brain, lung, prostate, placenta, retina and kidney), α-tocopherol. and while information about its non-hepatic roles is limited, αTTP is a cytosolic protein that is most highly expressed in some of the roles seem heterogeneous. In contrast to hepatocytes, where it traffics α-tocopherol and, with lower α-tocopherol efflux in hepatocytes,20 overexpression in prostate

6 Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from

Figure 6 The α-tocopherol transferase protein (αTTP) gene is required for CO2-mediated protection of compliance in murine ventilator induced − − lung injury. High-stretch (injurious) mechanical ventilation results in decreased static compliance in αTTP+/+ and αTTP / mice. Hypercapnia is protective (ie, reduces the injury) in αTTP+/+ (A), but not in αTTP−/− (B) mice (*p=0.001, t test). KO, knockout; WT, wild type. cells promotes accumulation of α-tocopherol,21 indicating mechanisms by direct application of tocopherols, such as aero- tissue-specific functions. In addition, human mutations in the solisation or incorporation into surfactant, might enhance their αTTP gene cause progressive ataxia and vitamin E deficiency effects. In addition, recent data indicate that metabolites, for (‘AVED’). example, γ-tocopherol or tocotrienols, may prove more Surprisingly, we did not demonstrate a role for αTTP against efficacious.24 oxidative stress in VILI, in terms of superoxide (figure 10A) or Beyond neutralisation of oxygen-derived or nitrogen- lipid peroxidation (ie, 8-Iso PGF2α, see online supplementary derived radicals, vitamin E can regulate eicosanoid metabol- table S4). This may be because of higher constitutive NRF2 anti- ism.17 Eicosanoids, including prostaglandins, thromboxanes, oxidant expression in αTTP colony mice versus C57BL/6J mice leukotrienes and HETEs, are an important network of signal- (figure 9C, D). Indeed, an increase in constitutive expression of ling molecules that regulate inflammatory responses in critical NRF-2-responsive genes in αTTP KO mice has been previously illness. For example, cyclic stretch of lung epithelial cells noted22 and may reflect an adaptation to lower levels of (mimicking dyspnoea or mechanical ventilation) activates 31 α-tocopherol. Our observation that WT sibs also display eleva- cPLA2, thereby providing free arachidonic acid for eicosa- tion may be due to copy insufficiency during development in a noid biosynthesis. α-Tocopherol modulates eicosanoid synthe- heterozygote mother. It is possible that induction of αTTP by sis through multiple means, such as preventing the oxidation hypercapnia in other strains (eg, C57Bl/6J) or species reduces of lipid precursors, modulating mediators derived from oxidative stress. 12-LOX and 12/15-LOX,32 and specific inhibition of There is nevertheless considerable evidence that vitamin E 5-LOX.17 Among a panel of arachidonate products, we identi-

(including α, β, γ and δ tocopherols and tocotrienols) modulates fied significant reduction in LTB4 and CysLTs (and a trend to http://thorax.bmj.com/ the innate immune response in lung. α-Tocopherol suppresses decreased 5-HETE) resulting from hypercapnic induction of inflammatory responses and lipopolysaccharide (LPS)-induced αTTP. Hypercapnia did not influence levels of other eicosa- oxidative injury in lung epithelial cells,23 and multiple formula- noids, including COX-derived prostaglandins, products of tions of vitamin E protect in lung injury models.24 For example, other LOX enzymes (12-HETE or 15-HETE) or in rodent models of LPS-induced lung injury, intraperitoneal non-enzymatic oxidation products of arachidonic acid α-tocopherol decreased lung oedema and neutrophil transmigra- (9-HETE, 11-HETE) (see online supplementary table S4). tion without affecting cytokine levels or NFκB activation,25 Together these data indicate that in our model, hypercapnia while enteral γ-tocopherol (a more powerful antioxidant than increased expression of αTTP; this increased accumulation on September 30, 2021 by guest. Protected copyright. α-tocopherol) decreased neutrophils and the key inflammatory and/or trafficking of α-tocopherol, leading in turn to specific 26 mediators MIP-2, CINC-1 and PGE2. A small human study inhibition of 5-LOX, and lower levels of leukotrienes. demonstrated that oral γ-tocopherol reduced sputum neutrophil Such lowering of leukotriene levels is a plausible downstream numbers following intranasal LPS.27 mechanism for protection against lung injury in the current However, important gaps exist in our understanding of the experiments. LTB4 is a potent PMN chemoattractant, providing protective effects of α-tocopherol: supplementation protects a key signal for transmigration into the airway mediated by spe- against lung injury in murine bacterial pneumonia cific G-protein coupled receptors (BLT1, BLT2), and CysLTs (Streptococcus pneumoniae), reducing mortality, bacterial load, increase vascular permeability and modulate vascular smooth and neutrophil transepithelial migration.28 In addition, while muscle tone via CysLT1 and CysLT2 receptors. A small clinical α-tocopherol protects against hyperoxic lung injury through study has demonstrated that plasma levels of leukotrienes were antioxidant mechanisms,29 the mechanisms of protection in elevated in patients with ARDS early in their illness, and early other lung injury (eg, sepsis) is unclear. LTB4 levels predicted mortality from ARDS.33 Most clinical studies of α-tocopherol supplementation show In other models of lung injury, antagonism of 5-LOX is pro- minimal or no benefit in patients who have adequate dietary tective; for example, pharmacological inhibition and gene intake,24 including the critically ill.30 An important limitation knockout of 5-LOX protect against murine VILI,18 and in not addressed in other studies is intracellular accumulation and murine sepsis KO of the 5-LOX gene (or inhibition of the trafficking of α-tocopherol in non-hepatic tissue. Increased enzyme) provided more protection than use of leukotriene αTTP expression could overcome such a limitation, although receptor antagonists (eg, Montelukast).34 Antagonism of LTB4 achieving increased expression would be a challenge. is also effective in lung injury caused by haemorrhagic shock35 Alternatively, bypassing limited endogenous transport or in carrageenan-induced pleurisy.36

Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 7 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from http://thorax.bmj.com/ on September 30, 2021 by guest. Protected copyright.

Figure 7 CO2-mediated protection and impact of α-tocopherol transferase protein (αTTP) on biochemical injury markers. Tissue levels of myeloperoxidase (MPO) activity (A) and TNFα mRNA (B) are significantly less after 3 hours of injurious ventilation with hypercapnia versus normocapnia (ie, hypercapnia protective) in αTTP+/+ (left panels) mice, but not in αTTP−/− (right panels) mice. Trends for these patterns − − (hypercapnic protection in αTTP+/+, not in αTTP / ) were preserved for bronchoalveolar lavage levels of monocyte chemoattractant protein 1 (MCP-1; C), keratinocyte chemoattractant (KC; D), and interleukin 6 (IL-6; E) (*p<0.05 vs normocapnia, t test). Data from non-ventilated mice are presented for illustrative purposes but excluded from statistical analysis. KO, knockout; WT, wild type.

8 Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from

Figure 8 Effects of mechanical ventilation and hypercapnia on lung α-tocopherol transferase protein (αTTP) mRNA expression and α-tocopherol levels. Hypercapnia induced expression of αTTP mRNA in lung tissue of αTTP+/+ mice following high-stretch ventilation (Mann–Whitney) (A). Linear regression demonstrates the correlation between expression of αTTP mRNA and CO2 protective effect (regression expressed±95% CIs; R=−0.50, p<0.05) (B). Lung tissue α-tocopherol (α-T) levels in αTTP+/+ mice are increased by hypercapnia during ventilation (t test) (C), and are 20–50 fold less in αTTP−/− mice (D) (*p<0.05 vs normocapnia). Data from (n=2–4) non-ventilated mice are presented for illustrative purposes but excluded from statistical analysis. KO, knockout; WT, wild type.

Figure 9 Antioxidant response http://thorax.bmj.com/ pathway in α-tocopherol transferase − − protein (αTTP+/+) and αTTP / murine model of ventilator-induced lung injury, effect of hypercapnia. αTTP+/+ − − and αTTP / mice expressed higher constitutive levels of mRNA for NRF2 target genes heme oxygenase (HMOX1) (A) and glutathione-S-transferase (GSTA1) (B) on September 30, 2021 by guest. Protected copyright. than C57BL/6J mice (§p<0.05 vs C57BL/6J, ANOVA on RANKS). Expression of HMOX1 (C) and GSTA1 (D) during injurious mechanical ventilation is similar in αTTP+/+and − − αTTP / mouse lungs. In non-ventilated mice, constitutive levels of HMOX1 and GSTA1 were higher in − − αTTP / versus αTTP+/+ mice (*p<0.01 vs αTTP+/+, two-way ANOVA). Both genes were similarly induced by VILI and this effect was abrogated by hypercapnia (†p<0.05 vs all other ventilation groups). KO, knockout; WT, wild type.

Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 9 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from http://thorax.bmj.com/ on September 30, 2021 by guest. Protected copyright.

Figure 10 Hypercapnia-induced α tocopherol transfer protein (αTTP) attenuates leukotriene synthesis, not reactive oxygen species production, in murine ventilator-induced lung injury. Relative levels of superoxide in αTTP+/+ and αTTP−/− mouse lungs were measured as 2-hydroxy-ethidium (2-OH-Et, arbitrary units) in mice injected with hydroethidine and subjected to 3 hours of injurious ventilation (A). There was no significant − − difference in superoxide levels between normocapnia and hypercapnia in αTTP / or αTTP+/+ animals (Mann–Whitney). Hypercapnia suppressed levels of leukotriene B4 (LTB4, t test) and cysteinyl leukotrienes (CysLTs, Mann–Whitney) in the bronchoalveolar fluid from αTTP+/+ mice subjected to − − injurious ventilation, but had no effect on leukotriene levels in αTTP / mice (B and C). Hypercapnia did not alter arachidonic acid levels in bronchoalveolar fluid from mice of either genotype (D) (*p<0.05 vs normocapnia). KO, knockout; WT, wild type.

10 Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from Despite preclinical success in experimental lung injury, Provenance and peer review Not commissioned; externally peer reviewed. caution is required in therapeutic targeting of leukotrienes. Data sharing statement Primary microarray datasets are accessible through NCBI Many patients with lung injury have concomitant sepsis, and GEO (series accession number GSE86229). reduced leukotriene function could impair pathogen clearance. In addition, 5-LOX inhibition could divert arachidonic acid via REFERENCES alternative pathways (eg, COX), thereby altering the balance of 1 ARDS Network. Ventilation with lower tidal volumes as compared with traditional eicosanoid signalling; in later stages this could impair produc- tidal volumes for acute lung injury and the acute respiratory distress syndrome. 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Although the role 19 Kono N, Arai H. Intracellular transport of fat-soluble vitamins A and E. Traffic of αTTP in lung tissue has received little attention, our data 2015;16:19–34. showing induction in multiple mouse models suggest broad 20 Qian J, Morley S, Wilson K, et al. Intracellular trafficking of vitamin E in – applicability, and the correlation with lung protection is evi- hepatocytes: the role of tocopherol transfer protein. J Lipid Res 2005;46:2072 82. 21 Morley S, Thakur V, Danielpour D, et al. Tocopherol transfer protein sensitizes dence of an induced, protective pathway. Targeted interventions prostate cancer cells to vitamin E. J Biol Chem 2010;285:35578–89. such as aerosolisation of vitamin E formulations into the lung, 22 Gohil K, Oommen S, Vasu VT, et al. Tocopherol transfer protein deficiency modifies or specific inhibition of LTA4H, are related to new approaches nuclear receptor transcriptional networks in lungs: modulation by cigarette smoke in to protecting the injured lung. vivo. Mol Aspects Med 2007;28:453–80. 23 Ekstrand-Hammarström B, Osterlund C, Lilliehöök B, et al. Vitamin E Acknowledgements This study was supported by Canadian Institutes of Health down-modulates mitogen-activated protein kinases, nuclear factor-kappaB and fl – Research Operating Grant to B.P.K. (FRN 69006). BAL mediator measurements were in ammatory responses in lung epithelial cells. Clin Exp Immunol 2007;147:359 69. fl performed by the Analytical Facility for Bioactive Molecules of The Centre for the 24 Jiang Q. Natural forms of vitamin E: metabolism, antioxidant, and anti-in ammatory Study of Complex Childhood Diseases, The Hospital for Sick Children, Toronto, activities and their role in disease prevention and therapy. Free Radic Biol Med – Canada. 2014;72:76 90. 25 Rocksén D, Ekstrand-Hammarström B, Johansson L, et al. Vitamin E reduces Contributors GO conceived the study, conducted the study, and analysed the transendothelial migration of neutrophils and prevents lung injury in endotoxin-induced data. DE, HA and HH participated in study design, conducted experiments, analysed airway inflammation. Am J Respir Cell Mol Biol 2003;28:199–207. data. HB, MP and BPK participated in study design, data analysis and interpretation. 26 Wagner JG, Birmingham NP, Jackson-Humbles D, et al. Supplementation with All authors participated in drafting or critically revising the manuscript and approved gamma-tocopherol attenuates endotoxin-induced airway neutrophil and mucous cell the final version. responses in rats. Free Radic Biol Med 2014;68C:101–9. Funding Institute of Circulatory and Respiratory Health (FRN 69006). 27 Hernandez ML, Wagner JG, Kala A, et al. Vitamin E, γ-tocopherol, reduces airway neutrophil recruitment after inhaled endotoxin challenge in rats and in healthy Competing interests None declared. volunteers. Free Radic Biol Med 2013;60:56–62.

Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501 11 Respiratory physiology Thorax: first published as 10.1136/thoraxjnl-2016-209501 on 3 February 2017. Downloaded from 28 Bou Ghanem EN, Clark S, Du X, et al. The α-tocopherol form of vitamin E reverses 34 Monteiro AP, Soledade E, Pinheiro CS, et al. Pivotal role of the 5-lipoxygenase age-associated susceptibility to streptococcus pneumoniae lung infection by pathway in lung injury after experimental sepsis. Am J Respir Cell Mol Biol modulating pulmonary neutrophil recruitment. J Immunol 2015;194:1090–9. 2014;50:87–95. 29 Yamaoka S, Kim HS, Ogihara T, et al. Severe vitamin E deficiency exacerbates acute 35 Eun JC, Moore EE, Mauchley DC, et al. The 5-lipoxygenase pathway is required for hyperoxic lung injury associated with increased oxidative stress and inflammation. acute lung injury following hemorrhagic shock. Shock 2012;37:599–604. Free Radic Res 2008;42:602–12. 36 Rossi A, Pergola C, Koeberle A, et al. The 5-lipoxygenase inhibitor, zileuton, 30 Howe KP, Clochesy JM, Goldstein LS, et al. Mechanical ventilation antioxidant trial. suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in Am J Crit Care 2015;24:440–5. macrophages. Br J Pharmacol 2010;161:555–70. 31 Copland IB, Reynaud D, Pace-Asciak C, et al. Mechanotransduction of 37 Li XJ, Fu HY, Yi WJ, et al. Dual role of leukotriene B4 receptor type 1 in stretch-induced prostanoid release by fetal lung epithelial cells. Am J Physiol Lung experimental sepsis. J Surg Res 2015;193:902–8. Cell Mol Physiol 2006;291:L487–95. 38 Stsiapanava A, Olsson U, Wan M, et al. Binding of Pro-Gly-Pro at the active site of 32 Lebold KM, Traber MG. Interactions between α-tocopherol, polyunsaturated fatty leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase acids, and lipoxygenases during embryogenesis. Free Radic Biol Med selective inhibitor. Proc Natl Acad Sci USA 2014;111:4227–32. 2014;66:13–19. 39 Koh M, Takitani K, Miyazaki H, et al. Liver X receptor up-regulates α-tocopherol 33 Amat M, Barcons M, Mancebo J, et al. Evolution of leukotriene B4, peptide transfer protein expression and α-tocopherol status. J Nutr Biochem 2013;24:2158–67. leukotrienes, and interleukin-8 plasma concentrations in patients at risk of acute 40 Ulatowski L, Dreussi C, Noy N, et al. Expression of the α-tocopherol transfer protein respiratory distress syndrome and with acute respiratory distress syndrome: mortality gene is regulated by oxidative stress and common single-nucleotide polymorphisms. prognostic study. Crit Care Med 2000;28:57–62. Free Radic Biol Med 2012;53:2318–26. http://thorax.bmj.com/ on September 30, 2021 by guest. Protected copyright.

12 Otulakowski G, et al. Thorax 2017;0:1–12. doi:10.1136/thoraxjnl-2016-209501